This invention pertains to the inhibition of herpes infections.
Herpes simplex virus type 1 (HSV-1) eye infections are very common in humans. Ocular herpes infections can have serious complications, including encephalitis, herpetic keratoconjunctivitis, and even blindness. Local immune responses to HSV-1 infection of the cornea include infiltration of mononuclear cells such as CD4+ and CD8+ T cells, macrophages, and neutrophils; the activation of natural killer cells; and the production of various cytokines. There are no successful HSV-1 vaccines commercially available today.
Next to trauma, the leading cause of blindness in the industrialized world is HSV infection. Approximately 6000 corneal transplants are conducted each year for patients with recurrent HSV infection. Ultimately, most of these patients lose their sight.
There are several subtypes of interferons. Type I interferons, such as IFN-xcex1 and IFN-xcex2 types, are naturally occurring hormones produced by the immune system and by non-immune cells in response to viral infections and other specific inducers. Endogenous type I interferons inhibit the replication of infectious virus in the host cells by destabilizing viral RNA, degrading viral nucleic acids, enhancing the expression of certain host proteins (e.g., MHC-I antigens), enhancing anti-viral effector mechanisms of the host immune system, and other anti-viral mechanisms. IFN-xcex1 has been used against established tumors and chronic viral infections (e.g., hepatitis B and C virus, AIDS-associated Kaposi""s sarcoma). Herpes simplex virus type I (xe2x80x9cHSV-1xe2x80x9d) is known to be sensitive to the anti-viral effects of IFN-xcex1.
D. Carr et al., xe2x80x9cAstrocyte-targeted expression of IFN-xcex11 protects mice from acute ocular herpes simplex virus type I infection,xe2x80x9d J. Immunol., vol. 161, pp. 4859-4865 (1998)(not admitted to be prior art) discloses that transgenic mice having a gene encoding IFN-xcex11 in the germline, where the gene is under the control of an astrocyte-specific promoter, were more resistant to HSV-1 infection than were non-transgenic control mice.
Some gene therapy efforts have used vectors encoding cytokine genes. Following in vivo transfection with a gene therapy vector, host cells take up plasmid DNA encoding the gene of interest. In situ expression of the transgene either antagonizes the microbial infection or reduces the destructive inflammatory process associated with the infection.
M. Dahesia et al., xe2x80x9cSuppression of ongoing ocular inflammatory disease by topical administration of plasmid DNA encoding IL-10, xe2x80x9d J. Immunol., vol. 159, pp. 1945-1952 (1997) disclosed that topical administration to the cornea of plasmid DNA encoding IL-10 reduced the incidence of herpetic stromal keratitis in mice; however, it was reported that similar effects were not seen with DNA encoding IL-2 and GM-CSF, the only other cytokine DNAs that had been tested to date.
W. Yeow et al., xe2x80x9cAntiviral activities of individual murine IFN-xcex1 subtypes in vivo: intramuscular injection of IFN expression constructs reduces cytomegalovirus replication,xe2x80x9d J. Immunol, vol. 160, pp. 2932-2939 (1998) discloses the intramuscular injection of naked DNA plasmids coding for IFN-xcex11, IFN-xcex14, or IFN-xcex19 subtypes into the tibialis anterior muscles of mice, followed by challenge with murine cytomegalovirus. Mice injected with the IFN-xcex1 plasmids showed lower CMV titers than did controls, with the lowest titers reported for the IFN-xcex11 DNA treatment.
S. Chun et al., xe2x80x9cModulation of viral immunoinflammatory responses with cytokine DNA administered by different routes,xe2x80x9d J. Virol., vol. 72, pp. 5545-5551 (1998) discloses that the systemic or topical administration of IL4 and IL-10 DNA, but not IL-2 or IFN-xcex3 DNA, suppressed HSV-specific, delayed-type hypersensitivity in mice. Inflammatory lesions associated with corneal HSV infection were suppressed by administering IL4 and IL-10 DNA to nasal mucosa or ocular surfaces.
H. Horton et al., xe2x80x9cA gene therapy for cancer using intramuscular injection of plasmid DNA encoding interferon xcex1,xe2x80x9d Proc. Natl. Acad. Sci. USA, vol. 96, pp. 1553-1558 (1999) discloses that the intramuscular injection of a plasmid encoding murine interferon-xcex1 in mice had significant anti-tumor effects.
I have discovered that the topical administration of a plasmid encoding interferon xcex11(IFN-xcex11) to the cornea has potent anti-herpes activity. The expression of the IFNxcex11 transgene in the corneas of infected individuals is transient and short-lived. The plasmid protected mice from HSV-1-induced encephalitis in a dose- and time-dependent manner when applied to the cornea prior to infection. The protective effect was mediated through the expression of the transgene rather than exposure to plasmid DNA, as indicated in experiments using neutralizing antibody to IFN-xcex1/xcex2. Gene therapy by delivery of a transgene in a plasmid should be qualitatively safer than the use of virus vectors, due to the lower potential for adverse effects.
The novel method may be used to treat or prevent herpes infections not only in the eye, but also in other portions of the oropharyngeal region, and in other mucous membranes including the genitalia. Patients presenting with acute or reactivated HSV-1 or other herpes infections are topically treated with DNA (e.g., a plasmid) encoding a type I interferon such as interferon-xcex11 (xe2x80x9cIFN-xcex11xe2x80x9d), where the DNA is not designed to integrate into the genome, but is placed under the control of a promoter that will cause expression of the interferon while the DNA persists in the cells that take it up.
Viral replication may be inhibited by targeting constitutive but transient expression of type I IFN genes to cells infected by herpes virus. In particular, the experimental results reported here show that the transgene blocks viral replication by antagonizing the expression of immediate early and early viral genes that are necessary for productive infection. The transgene up-regulates the expression of MHC-I antigen by the transfected tissue, facilitating the presentation of viral antigen to the immune system, and subsequently targeting infected cells for destruction by immune effector cells.
The novel method may be used either independently, or in conjunction with other antiviral therapies, such as acyclovir or ganciclovir.
By limiting viral replication, the novel treatment reduces the initial spread of virus, and thereby also reduces the establishment of latent infections. Typically, most morbidity from an HSV-1 infection occurs when latently-infected cells (neurons that are infected with the virus, but that for a time do not express any viral antigens or produce infectious virus) reactivate in response to environmental cues. Tissue destruction then results as host immune cells respond to the reactivated virus and destroy the infected cells. Patients with recurrent reactivation of latent HSV-1 can, for example, lose their sight or experience various autoimmune diseases indirectly triggered by the virus. HSV reactivation plagues approximately 20% of infected individuals, some of which become serious cases of reactivation.
The novel method of treating HSV infection may be used to control ocular and other HSV infections. The plasmid construct (DNA only) is placed in a sterile solution with appropriate buffers to optimize in situ transfection of target tissue. For example, we have placed the plasmid in phosphate buffered saline (pH 7.4) at a concentration of 33.3 xcexcg/xcexcl. We have found that the plasmid construct is stable in this storage medium for about 10 days. It then begins to degrade, with little or no efficacy remaining after 21 days.
DNA purification resins such as are known in the art are used to minimize endotoxin contamination of the plasmid DNA. The solution is applied to the target tissue (e.g., the eyes) topically, typicallyxcx9c3 xcexcl volume. As discussed below, the results of initial animal tests were quite promising. This construct or analogous constructs will therefore be tested in humans for efficacy against HSV-1 in compliance with pertinent laws and regulations.
This invention provides a low-cost, non-invasive treatment for HSV infection that may either be administered by physicians or other health care personnel, or self-administered by patients. An important benefit of the present invention is its strong inhibition of latent infections if applied during the acute phase of infection. Reactivation should therefore be greatly reduced. The application of exogenous interferons has previously been shown to be non-protective against re-exposure to virus, since it does not lead to the mounting of an active immune response. However, the novel transgene approach leads to the mounting of a substantial humoral immune response, a response that is essentially identical to the immune response shown by individuals who survive the initial infection and mount a significant adaptive immune response to the virus.